Simultaneous determinations of oxygen uptake, the oxidation of NADPH and NADH, and ethylmorphine by liver microsomes isolated from normal, phenobarbital and pregnenolone 16 alpha-carbonitrile pretreated rats revealed that a part of oxidation of NADH results in the formation of an unstable active oxygen species, but does not result in the reduction of this species. These findings indicate that the role of cytochrome b5, is closely intertwined with the functional state NADPH-mediated P-450 system as a source of the second electron in drug metabolism and the formation of the active oxygen species.